1. Field of the Invention
The present invention relates to a method of isolating and purifying trehalose. Trehalose and its various hydrates are well known as cell activity-retaining agents, cold-resisting agents, an anti-freezing agents, etc. in the fields of medicine, biochemistry and food science.
2. Description of the Prior Art
Trehalose has been isolated by extraction from dry yeast or the like; by enzymatic production and isolation, and by the culturing of microorganisms. Regarding the extraction of trehalose from dry yeast, methods have been reported wherein trehalose is extracted with ether or alcohol followed by the repeated crystallization of the extracted trehalose with alcohol and acetone (Science, 61 (1587), 570, 1925); wherein trehalose is extracted from yeast by treatment with NH.sub.2 SO.sub.4 and heavy metals (HgSO.sub.4 +Fe.sub.2 (SO.sub.4).sub.3) and a large amount of 95% alcohol is added to the extract to crystallize trehalose (Science, 82 (2131), 422, 1935); wherein trehalose is extracted from yeasts with alcohol, ionic substances are removed with ion-exchange resins (Amberlite IR-100, IR-4B), and a large amount of 95% alcohol is added to the extract to crystallize trehalose (J. Am. Chem. Soc., 72, 2059, 1950), etc. However, as these methods require a plurality of complicated steps for purification, they are not suitable for mass-production or for producing large amounts of trehalose. In addition, the purity of the trehalose obtained by the above-described methods is low, as is their yield.
The enzymatic production of trehalose has been described wherein maltose is treated with a maltose phosphorylase and a trehalose phosphorylase to produce trehalose, precipitates are removed from the enzymatically treated liquid, and the product is purified by treatment with an anion-exchange resin (Japanese Patent Application Laid-Open No. 58-216695). The trehalose-containing liquid purified by treatment with an anion-exchange resin is then applied to a borate-type anion-exchange resin so that the trehalose is adsorbed thereto, and the adsorbed trehalose is eluted and fractionated with a potassium borate solution. The obtained trehalose fraction is treated with a cation-exchange resin and concentrated, the concentrated liquid is distilled after adding a lower alcohol thereto so as to remove boric acid therefrom, and thereafter this concentrate is repeatedly crystallized with alcohol to obtain crystals of trehalose dihydrate.
Alternatively, sucrose is treated with an immobilized glycosyl transferase to produce paratinose, which is crystallized and separated, and the mother liquid containing by-product trehalose is treated with an anion-exchange resin of a mixed sulfite/bisulfite type and treated with a cation-exchange resin of the Ca-type to purify and isolate trehalose (Japanese Patent Application Laid-Open No. 4-131090). However, since these methods require purification or treatment with ion-exchange resins, they are not suitable for mass-production or for producing a large amount of trehalose. In addition, the purity of the trehalose as produced by such methods is low.
The production of trehalose by culturing microorganisms has been described where trehalose-producing bacteria of the genus Nocardia were cultured, the cells were removed from the culture liquid, the culture liquid was treated with methanol, insoluble substances were removed by filtration, and trehalose was isolated from the filtrate (Japanese Patent Laid-Open Application No. 50-154485). In the disclosed method, the filtrate is treated twice each with an anion-exchange resin and a cation-exchange resin and then subjected to gel filtration followed by adsorption to and elution from active charcoal, and the resulting elutate is crystallized from ethanol repeatedly to obtain crystals of trehalose.
Also known is a method of culturing fungi of the genus Rhizoctonia, Sklerotium or the like, followed by separating the cells from the culture liquid, triturating the cells, extracting them with an aqueous trichloroacetic acid solution, and isolating trehalose from the resulting extract (Japanese Patent Application Laid-Open No. 3-130084). In the disclosed method, the extract is first treated with chloroform and ether to remove lipids and trichloroacetic acid, there it is treated with an ion-exchange resin and dried to solid by evaporation. The solid is dissolved in acetonitrile or the like, and the solution is subjected to silica gel chromatography to isolate trehalose. Alternatively, the cells are removed from the culture liquid, the resulting supernatant is concentrated and then subjected to chromatography (Bio-Gel P-2), the trehalose fraction thus obtained is treated with Dowex 50 and again subjected to chromatography (Bio-Gel P-2), and finally dried to solid by evaporation to obtain a dry product of trehalose (Agric. Biol. Chem. 52 (3), 867-868, 1988). However, since these methods require various chromatographic processes, they are not suitable for mass-production or for producing a large amount of trehalose. In addition, the purification methods require plural steps.
As mentioned above, known processes for isolating trehalose generally contain an alcoholic crystallization step. However, the crystals of trehalose dihydrate obtained by alcoholic crystallization are problematic in that they are fine and have a low purity.